2 substituted datp derivatives as building blocks for polymerase catalyzed synthesis of dna modified in the minor groove

German Edition: DOI: 10.1002/ange.2016090072-Substituted dATP Derivatives as Building Blocks for Polymerase-Catalyzed Synthesis of DNA Modified in the Minor Groove Jn Matyasˇovsky´, Pav

German Edition: DOI: 10.1002/ange.201609007

2-Substituted dATP Derivatives as Building Blocks for

Polymerase-Catalyzed Synthesis of DNA Modified in the Minor Groove

Jn Matyasˇovsky´, Pavla Perlkov, Vincent Malnuit, Radek Pohl, and Michal Hocek*

Abstract: 2’-Deoxyadenosine triphosphate (dATP) derivatives

bearing diverse substituents (Cl, NH2, CH3, vinyl, ethynyl, and

phenyl) at position 2 were prepared and tested as substrates for

DNA polymerases The 2-phenyl-dATP was not a substrate for

DNA polymerases, but the dATPs bearing smaller substituents

were good substrates in primer-extension experiments,

produc-ing DNA substituted in the minor groove The vinyl-modified

DNA was applied in thiol–ene addition and the

ethynyl-modified DNA was applied in a CuAAC click reaction to form

DNA labelled with fluorescent dyes in the minor groove

Base-modified oligonucleotides (ONs) or DNA are widely

used as tools in chemical biology, diagnostics, or materials

science.[1]The modification is mostly attached to position 5 of

pyrimidines or position 7 of 7-deazapurines, not only because

it then points out into the major groove of DNA and thus does

not destabilize the duplex, but because in most cases, the

corresponding substituted 2’-deoxyribonucleoside

triphos-phates (dNTPs) are good substrates for DNA polymerases

and can be used in the polymerase-catalyzed synthesis of

modified DNA.[2, 3] Diverse modifications, including

fluoro-phores,[4] redox[5] or spin labels,[6] reactive groups for

con-jugations,[7] and biomolecules (e.g., oligonucleotides[8] or

proteins[9]), have been introduced into the major groove

through the enzymatic incorporation of modified nucleotides

and applied in different fields Modification or labelling of the

minor groove has mostly been reported with 2’- and

4’-sugar-modified derivatives.[10–13]2-Chloroadenine[14]and

2,6-diami-nopurine[15]dNTPs are the only minor-groove base-modified

nucleotides that have been reported as substrates for DNA

polymerases, whereas 2-arylamino-dATP derivatives were found to act as polymerase inhibitors.[16]The minor groove sites of the nucleobases are difficult to modify since they are crucial both for Watson–Crick base pairing and for key minor-groove interactions with DNA polymerase that are important for extension of the chain.[17]On the other hand, 2-ethynyl-pyridone-C-nucleotide incorporated into DNA[18] formed

a stable base pair with adenine, and 2-(imidazolylalkylami-no)purines in ONs also stabilized duplexes.[19] Because the possibility of minor-groove base labelling would be attractive for many prospective applications, for example, the mapping

of DNA–protein interactions, we envisaged that a small substituent at position 2 of a purine may not fully disturb the key H-bonding interactions with the opposite base and the polymerase, and we report herein the first enzymatic syn-thesis of minor-groove base-modified DNA

A series of six 2-substituted dATP derivatives bearing Cl,

NH2, CH3, vinyl, ethynyl and phenyl substituents (dRATPs) was designed to study the effect of substituents of different bulkiness at position 2 of adenine on polymerase-mediated incorporation While dCl

ATP[14, 20] and dNH2

ATP[15] were known, the others were prepared through triphosphoryla-tion[21]of the corresponding 2’-deoxy-ribonucleosides (dRAs, Scheme 1), which were synthesized through cross-coupling reactions of the 2-iodo-2’-deoxyadenosine (for details of the synthesis, see the Supporting Information)

The dRATPs were then tested as substrates for DNA polymerases in primer extension (PEX) experiments First,

we performed PEX in presence of KOD XL, Vent(exo-), or Bst DNA polymerase, using a 15-nt primer (prim248short) and 19-nt template (tempoligo1A) designed for the incorporation of one modified nucleotide, and the outcome was analyzed by denaturing polyacrylamide gel electrophoresis (PAGE) All three DNA polymerases (Figure 1 a and Figure S1 in the Supporting Information) incorporated the 2-substituted deox-yadenosine nucleotides, giving clean full-length DNA prod-ucts (DNA1RA) The only exception was the 2-phenyl derivative dPhATP, which apparently was not a substrate for DNA polymerases since almost no extension was observed

Then PEX was conducted using a longer 31-nt template (tempPrb4basII, which is modified with TINA at 3’-end to prevent non-templated incorporation,[22] Figure 1 b and Fig-ure S2) designed for the incorporation of 4RA modifications

Most of the modified dRATPs were good substrates, giving full-length products (DNA4R

A) Only the PEX product from ethynylated dEATP and KOD XL DNA polymerase was partially halted at the n 1 position (but Vent(exo-) and Bst DNA polymerases gave clean full-length products; see Fig-ure S2), while dPhATP did not give PEX with any of the tested DNA polymerases All of the PEX experiments (with all of

[*] J Matyasˇovsky´, Dr P Perlkov, Dr V Malnuit, Dr R Pohl,

Prof Dr M Hocek

Institute of Organic Chemistry and Biochemistry

Czech Academy of Sciences

Flemingovo nam 2, 16610 Prague 6 (Czech Republic)

E-mail: hocek@uochb.cas.cz

Homepage: http://www.uochb.cas.cz/hocekgroup

Prof Dr M Hocek

Department of Organic Chemistry, Faculty of Science

Charles University in Prague

Hlavova 8, 12843 Prague 2 (Czech Republic)

Supporting information and the ORCID identification number(s) for

the author(s) of this article can be found under http://dx.doi.org/10.

1002/anie.201609007.

 2016 The Authors Published by Wiley-VCH Verlag GmbH & Co.

KGaA This is an open access article under the terms of the Creative

Commons Attribution Non-Commercial NoDerivs License, which

permits use and distribution in any medium, provided the original

work is properly cited, the use is non-commercial, and no

modifications or adaptations are made.

the dRATPs and both templates) with KOD XL DNA

polymerase were repeated using biotinylated templates, and

the modified single-stranded oligonucleotides (ONs, ON1RA

or ON4RA) were isolated by magnetoseparation[23] and

analyzed by MALDI-TOF, which confirmed their identity

(Table S2 in the Supporting Information)

To further quantify the substrate activities of the modified

dRATPs, we conducted a simple kinetic analysis of

single-nucleotide extension using KOD XL DNA polymerase and

temp1A_term (Figure 1 c) and compared the conversion as

a function of time to that observed with natural dATP The

rate of extensions when using the smaller derivatives dClATP,

dNH2ATP, or dMeATP were comparable to the rate with

natural dATP, whereas PEX with the bulkier dVATP and

dEATP took approximately 2 min to reach completion To

study the influence of the 2-modifications on the base pairing

and duplex stability, we measured the denaturing

temper-atures of all of the PEX products (Table 1) Except for

2,6-diaminopurine, which stabilized the dsDNA due to an

additional H-bond with T, all of the other modifications

destabilized the duplexes

With the shorter (DNA1VA or DNA1EA) and longer

(DNA4VA or DNA4EA) dsDNA containing one or four

2-vinyl- or 2-ethynyladenine modifications in hand, we tested

whether they could be used for post-synthetic minor-groove

fluorescence labelling The vinyl group was envisaged for use

in the thiol–ene reaction (Scheme 1 c),[24]whereas the ethynyl group was envisaged for use in Cu-catalyzed alkyne–azide cycloaddition (CuAAC; Scheme 1 d).[25]We selected coumar-inemethylthiol (CM-SH)[26] and the commercially available azide-conjugated Cy3 (Cy3-N3) as model reagents The thiol– ene reactions of DNA1VA or DNA4VA with CM-SH pro-ceeded in 3 days at 37 8C without UV irradiation to give approximately 60 % conversion (based on PAGE analysis, Figure 2 d) to blue-fluorescent conjugates (DNA1CMA or DNA4CMA; Figure 2 a,c) In this case, UV irradiation did not help, owing to bleaching of the fluorophore The CuAAC reactions of DNA1EA or DNA4EA with Cy3-N3 proceeded smoothly at 37 8C in the presence of CuBr, sodium ascorbate, and tris(benzyltriazolylmethyl)amine (TBTA), quantitatively providing the red-fluorescent Cy3-triazole-modified DNA (DNA1Cy3A or DNA4Cy3A; Figure 2 b,e,f) Interestingly, the

Figure 1 a, b) Denaturating PAGE of PEX experiments in presence of KOD XL with temp oligo1A (a) or temp Prb4basII -TINA (b) P: primer, + : products of PEX with natural dNTPs, A : products of PEX with dTTP, dCTP, and dGTP; R A: products of PEX with dTTP, dCTP, dGTP, and functionalized d R ATP c) PAGE analyses of the kinetics of single-nucleotide extension experiments with temp 1A term , KOD XL, and d R ATP compared to natural dATP Time intervals are given in minutes.

Table 1: Denaturing temperatures of modified DNA duplexes.

DNA1 Cl

DNA1 NH2

DNA1 Me

DNA1 V

DNA1 E

DNA1 Cy3

[a] DT m = (T m mod T m natur )/n mod

Scheme 1 a) Synthesis of 2-substituted dATP derivatives b) PEX

incor-poration of the modified nucleotides into DNA c, d) Post-synthetic

minor-groove fluorescent labelling by thiol–ene (c) or CuAAC (d)

reactions.

triazole-linked duplex was more stable than the starting

ethynyl-modified DNA (Table 1)

In conclusion, we found that not only 2-chloro-[14]and

2-aminoadenine[15]dNTPs, but also dATP derivatives bearing

smaller C substituents at position 2 (CH3, vinyl, and ethynyl)

are good substrates for DNA polymerases and can be used for

the enzymatic synthesis of base-modified DNA bearing

substituents in the minor groove Conversely, the phenyl

group is too bulky because the corresponding dPhATP was not

a substrate for any tested DNA polymerase The

minor-groove vinyl- or ethynyl-modified DNAs can be

post-syn-thetically labelled through thiol–ene or CuAAC reactions

with thiols or azides, which was exemplified by fluorescent

labelling with coumarine or Cy3 To the best of our

knowl-edge, this is the first example of polymerase-catalyzed

synthesis of DNA modified at the minor groove sites of

nucleobases, and it paves the way for other minor-groove

nucleobase modifications and conjugations,[27]which could be

useful in applications in chemical biology or imaging Studies

along these lines are underway

Acknowledgements

This work was supported by the Czech Academy of Sciences

(RVO: 61388963 and the Praemium Academiae award to M

H.), by the Czech Science Foundation (14-04289S to M.H.) by

the Marie Sklodowska-Curie Innovative Training Network (ITN) Click Gene (H2020-MSCA-ITN-2014-642023 to J.M.)

Keywords: bioconjugation · DNA modification · DNA polymerase · nucleotides · fluorescent labelling

[1] Modified Nucleic Acids, Nucleic Acids and Molecular Biology Series, Vol 31 (Eds.: K Nakatani, Y Tor), Springer, Berlin, 2016,

pp 1 – 276.

[2] Reviews: a) A Hottin, A Marx, Acc Chem Res 2016, 49, 418 – 427; b) M Hollenstein, Molecules 2012, 17, 13569 – 13591; c) M.

Hocek, J Org Chem 2014, 79, 9914 – 9921; d) M Kuwahara, N.

Sugimoto, Molecules 2010, 15, 5423 – 5444.

[3] Recent examples: a) S Obeid, A Baccaro, W Welte, K.

Diederichs, A Marx, Proc Natl Acad Sci USA 2010, 107,

21327 – 21331; b) K Bergen, A L Steck, S Strtt, A Baccaro,

W Welte, K Diederichs, A Marx, J Am Chem Soc 2012, 134,

11840 – 11843; c) P Kielkowski, J Fanfrlk, M Hocek, Angew.

Chem Int Ed 2014, 53, 7552 – 7555; Angew Chem 2014, 126,

7682 – 7685.

[4] Recent examples: a) D Dziuba, P Jurkiewicz, M Cebecauer, M.

Hof, M Hocek, Angew Chem Int Ed 2016, 55, 174 – 178;

Angew Chem 2016, 128, 182 – 186; b) D Dziuba, P Pospsˇil, J.

Matyasˇovsky´, J Brynda, D Nachtigallov, L Rulsˇek, R Pohl,

M Hof, M Hocek, Chem Sci 2016, 7, 5775 – 5785; c) M.

Tokugawa, Y Masaki, J C Canggadibrata, K Kaneko, T.

Shiozawa, T Kanamori, M Grøtli, L M Wilhelmsson, M.

Sekine, K Seio, Chem Commun 2016, 52, 3809 – 3812.

[5] a) J Balintov, M Plucnara, P Vidlkov, R Pohl, L Havran,

M Fojta, M Hocek, Chem Eur J 2013, 19, 12720 – 12731; b) J.

Balintov, J Sˇpacˇek, R Pohl, M Brzdov, L Havran, M Fojta,

M Hocek, Chem Sci 2015, 6, 575 – 587.

[6] S Obeid, M Yulikov, G Jeschke, A Marx, Angew Chem Int.

Ed 2008, 47, 6782 – 6785; Angew Chem 2008, 120, 6886 – 6890.

[7] a) S H Weisbrod, A Marx, Chem Commun 2007, 1828 – 1830;

b) V Borsenberger, S Howorka, Nucleic Acids Res 2009, 37,

1477 – 1485; c) J Schoch, M Wiessler, A Jschke, J Am Chem.

Soc 2010, 132, 8846 – 8847; d) K Gutsmiedl, D Fazio, T Carell, Chem Eur J 2010, 16, 6877 – 6883; e) V Raindlov, R Pohl, M.

Hocek, Chem Eur J 2012, 18, 4080 – 4087; f) J Dadov, P.

Orsg, R Pohl, M Brzdov, M Fojta, M Hocek, Angew.

Chem Int Ed 2013, 52, 10515 – 10518; Angew Chem 2013, 125,

10709 – 10712; g) S Arndt, H A Wagenknecht, Angew Chem.

Int Ed 2014, 53, 14580 – 14582; Angew Chem 2014, 126, 14808 – 14811; h) K Wang, D Wang, K Ji, W Chen, Y Zheng, C Dai, B.

Wang, Org Biomol Chem 2015, 13, 909 – 915; i) X Ren, A H.

El-Sagheer, T Brown, Nucleic Acids Res 2016, 44, e79; j) A.

Olszewska, R Pohl, M Brzdov, M Fojta, M Hocek, Bioconjugate Chem 2016, 27, 2089 – 2094.

[8] A Baccaro, A L Steck, A Marx, Angew Chem Int Ed 2012,

51, 254 – 257; Angew Chem 2012, 124, 260 – 263.

[9] M Welter, D Verga, A Marx, Angew Chem Int Ed 2016, 55,

10131 – 10135; Angew Chem 2016, 128, 10286 – 10290.

[10] a) M M Rubner, C Holzhauser, P R Bohlnder, H A Wagen-knecht, Chem Eur J 2012, 18, 1299 – 1302; b) S Berndl, N.

Herzig, P Kele, D Lachmann, X Li, O S Wolfbeis, H A.

Wagenknecht, Bioconjugate Chem 2009, 20, 558 – 564.

[11] U Wenge, T Ehrenschwender, H A Wagenknecht, Bioconju-gate Chem 2013, 24, 301 – 304.

[12] a) L H Lauridsen, J A Rothnagel, R N Veedu, ChemBio-Chem 2012, 13, 19 – 25; b) M Kuwahara, S Obika, J Nagashima,

Y Ohta, Y Suto, H Ozaki, H Sawai, T Imanishi, Nucleic Acids Res 2008, 36, 4257 – 4265; c) T Chen, N Hongdilokkul, Z Liu,

Figure 2 a) Normalized emission spectra of DNA1 V

A and DNA4 V

A compared to non-modified DNA1A or DNA4A before and after thiol–

ene reaction with CM-SH b) Normalized emission spectra of DNA1 E

A and DNA4 E

A compared to non-modified DNA1A or DNA4A before and

after CuAAC reaction with Cy3-N 3 c) A photograph of vials containing

CM-linked DNA under UV irradiation (365 nm) compared to

non-modified DNA treated with the same reagent d) PAGE analysis of PEX

product ON1 V

A and the product of subsequent thiol–ene reaction,

ON1 CM

A e) A photograph of vials containing Cy3-linked DNA (in H 2 O/

glycerol) under UV irradiation (365 nm) compared to non-modified

DNA treated with the same reagent f) PAGE analysis of PEX product

ON1 E

A and the product of subsequent CuAAC reaction, ON1 Cy3

A.

R Adhikary, S S Tsuen, F E Romesberg, Nat Chem 2016, 8,

556 – 562.

[13] A Marx, M P MacWilliams, T A Bickle, U Schwitter, B Giese,

J Am Chem Soc 1997, 119, 1131 – 1132.

[14] W B Parker, S C Shaddix, C.-H Chang, E L White, L M.

Rose, R W Brockman, A T Shortnacy, J A Montgomery, J A.

Secrist III, L L Bennett, Cancer Res 1991, 51, 2386 – 2394.

[15] a) A Chollet, E Kawashima, Nucleic Acids Res 1988, 16, 305 –

317; b) I V Kutyavin, Biochemistry 2008, 47, 13666 – 13673.

[16] a) N N Khan, G E Wright, L W Dudycz, N C Brown, Nucleic

Acids Res 1985, 13, 6331 – 6342; b) B Zdrazil, A Schwanke, B.

Schmitz, M Schfer-Korting, H D Hçltje, J Enzyme Inhib.

Med Chem 2011, 26, 270 – 279.

[17] S Matsuda, A M Leconte, F E Romesberg, J Am Chem Soc.

2007, 129, 5551 – 5557.

[18] M Minuth, C Richert, Angew Chem Int Ed 2013, 52, 10874 –

10877; Angew Chem 2013, 125, 11074 – 11077.

[19] K S Ramasamy, M Zounes, C Gonzales, S M Freier, E A.

Lesnik, L L Cummins, R H Griffey, B P Monia, P D Cook,

Tetrahedron Lett 1994, 35, 215 – 218.

[20] N D’Ambrosi, S Constanzi, D F Angelini, R Volpini, G.

Sancesario, G Cristalli, C Volonte, Biochem Pharmacol 2004,

67, 621 – 630.

[21] T Kovcs, L tvçs, Tetrahedron Lett 1988, 29, 4525 – 4528 [22] P Gixens-Gallardo, M Hocek, P Perlkov, Bioorg Med Chem Lett 2016, 26, 288 – 291.

[23] P Brzdilov, M Vrbel, R Pohl, H Pivonkov, L Havran, M Hocek, M Fojta, Chem Eur J 2007, 13, 9527 – 9533.

[24] a) C E Hoyle, C N Bowman, Angew Chem Int Ed 2010, 49,

1540 – 1573; Angew Chem 2010, 122, 1584 – 1617; b) C E Hoyle, A B Lowe, C N Bowman, Chem Soc Rev 2010, 39,

1355 – 1387.

[25] a) P M E Gramlich, C T Wirges, A Manetto, T Carell, Angew Chem Int Ed 2008, 47, 8350 – 8358; Angew Chem.

2008, 120, 8478 – 8487; b) A H El-Sagheer, T Brown, Chem Soc Rev 2010, 39, 1388 – 1405.

[26] S Akita, N Umezawa, T Higuchi, Org Lett 2005, 7, 5565 – 5568 [27] M Merkel, K Peewasan, S Arndt, D Ploschik, H A Wagen-knecht, ChemBioChem 2015, 16, 1541 – 1553.

Received: September 14, 2016 Published online: && &&, &&&&

DNA Modification

J Matyasˇovsky´, P Perlkov, V Malnuit,

R Pohl, M Hocek* &&&&—&&&&

2-Substituted dATP Derivatives as

Building Blocks for Polymerase-Catalyzed

Synthesis of DNA Modified in the Minor

Groove

Get into the (minor) groove: Enzymatic synthesis of minor-groove-modified DNA was achieved through the polymerase-catalyzed incorporation of 2-substituted 2’-deoxyadenosine nucleotides Postsyn-thetic minor-groove labelling was subse-quently carried out through thiol–ene or CuAAC reaction of the vinyl-modified or ethynyl-modified DNA, respectively